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G Nagel

Publications and source records attributed to G Nagel.

At least 91 records · Page 5Linked to original sources
PubMed1992

The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator.

Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined.

Adenosine Triphosphate↗
PubMed1992

CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells.

Neutrophil adherence to cytokine-activated endothelial cell (EC) monolayers depends on the expression of the endothelial leukocyte adhesion molecule-1 (ELAM-1). The ligand for ELAM-1 is the sialylated Lewis-x antigen (SLe(x)) structure. The selectin LAM-1 (or LECAM-1) has been described as one of the SLe(x)-presenting glycoproteins involved in neutrophil binding to ELAM-1. Other presenter molecules have not yet been described. Our data demonstrate that the carcinoembryonic antigen (CEA)-like surface molecules on neutrophils--known as the nonspecific cross-reacting antigens (NCAs)--are involved in neutrophil adherence to monolayers of IL-1-beta-activated EC. The NCAs are recognized by CD66 (NCA-160 and NCA-90) and CD67 (NCA-95). Because NCA-95 and NCA-90 have previously been found to be phosphatidylinositol (PI)-linked, paroxysmal nocturnal hemoglobinuria (PNH) neutrophils (which lack PI-linked surface proteins) were tested as well. PNH neutrophils showed a diminished binding to activated EC. CD66 (on PNH cells still recognizing the transmembrane NCA-160 form) still inhibited the adherence of PNH cells to IL-1-beta-activated EC, but to a limited extent. Soluble CEA(-related) antigens inhibited normal neutrophil adherence as well, whereas neutrophil transmigration was unaffected. Sialidase-treatment as well as CD66 preclearing abolished the inhibitory capacity of the CEA(-related) antigens. The binding of soluble CEA antigens to IL-1-beta-pretreated EC was blocked by anti-ELAM-1. These soluble antigens, as well as the neutrophil NCA-160 and NCA-90, both recognized by CD66 antibodies, presented the SLe(x) determinant. Together, these findings indicate that the CD66 antigens (i.e., NCA-160/NCA-90) function as presenter molecules of the SLe(x) oligosaccharide structures on neutrophils that bind to ELAM-1 on EC.

Antigens, CD↗
PubMed1992

Charge movements via the cardiac Na,K-ATPase.

The voltage dependence of transient and steady-state pump currents was examined in guinea pig ventricular myocytes to investigate mechanisms of charge translocation by the Na,K-ATPase. Na/K pump current was determined at approximately 36 degrees C as strophanthidin-sensitive whole-cell current in myocytes voltage clamped and internally dialyzed via wide tipped pipettes containing a pipette perfusion device. External Na ions diminished stationary pump current during forward Na/K cycling in a voltage dependent manner, the inhibition becoming stronger upon hyperpolarization. When Na,K-ATPase activity was restricted to Na translocation steps, stationary pump current was prevented but voltage pulses still elicited large transient pump currents which could be abolished by oligomycin B (> or = 2 micrograms/ml). The transients arose instantaneously on stepping the voltage, and decayed with voltage-dependent approximately single exponential time courses. The decay rates, and their high temperature sensitivity (approximately 200 s-1 at 0 mV at 36 degrees C; approximately 40 s-1 at 20 degrees C), suggest that the charge movements were limited by a conformational change associated with Na deocclusion. Those rates varied asymmetrically with voltage, changing little at positive voltages but increasing roughly exponentially with hyperpolarization (e-fold/approximately 80 mV). Lowering the extracellular [Na] ([Na]o) slowed the relaxation of charge movement at negative potentials but had little effect at positive potentials, and so shifted the rate constant-voltage curve to the left. The implied dependence on [Na]o of the backward rate constant governing pump charge movement accounts satisfactorily for the observed [Na]o sensitivity of stationary outward Na/K pump current, and indicates that the voltage-dependent step somehow involves the release of Na ions to the external medium. However, no strophanthidin-sensitive current was seen, at saturating external [K], when Na,K-ATPase activity was limited to K translocation steps by complete withdrawal of Na ions. But, at very low [Na]o, a weak negative slope appeared in the stationary pump current-voltage relationship at subsaturating, but not at saturating, external [K], indicating an increased apparent affinity for external K at more negative potentials. The results support the existence of a high field access channel through which extracellular Na and K ions must pass before interacting with their binding sites deep within the Na,K-ATPase molecule.

Adenosine Triphosphate↗
PubMed1991

CD66 identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members of the carcinoembryonic antigen family of adhesion molecules.

Preliminary results from the IVth Leucocyte Culture Conference have classified the monoclonal antibody (MoAb), YTH 71.3.2, as CD66. Two other MoAbs, YPC 2/12.1 and CE6/2D3.1, share a common cellular specificity, reacting with cells of the neutrophil series and colonic epithelium. The YTH 71.3.2 and CE6/2D3.1 MoAbs both recognize a similar CD66 defined epitope that is distinct from that identified by YPC 2/12.1. By Western blotting, these antibodies react with different molecular species from cells of different lineages. The antibodies identify 50- to 55-Kd, 80- to 100-Kd, and 130- to 200-Kd components present in a semi-purified carcinoembryonic antigen (CEA) preparation from colonic adenocarcinomas and a 90- to 130-Kd molecule from HL-60 cells. With the colonic cell line, LS174T, YPC2/12.1 stains diffuse bands of 160 to 200 Kd and 90 to 130 Kd with equal intensity, whereas the binding of CE6/2D3.1 and YTH 71.3.2 is biased toward the lower molecular weight set of molecules. Remarkably, all three antibodies recognize CEA-related molecules. Defined analyses using HeLa cells transfected with CEA, NCA(NCA-50/90), and CGM6(NCA-95) cDNAs show that the three MoAbs identify CEA to varying degrees. While YTH 71.3.2 and CE6/2D3.1 also bind to NCA-50/90, YPC 2/12.1 recognizes an epitope expressed by both the NCA-50/90 and NCA-95 molecular species.

Antibodies, Monoclonal↗
PubMed1991

Limited proteolysis of lactate dehydrogenase from porcine heart with trypsin: characterization and reactivation of the fragments.

The ternary complex formed by native lactate dehydrogenase (LDH) from porcine heart, NAD+ and sulfite, was digested with trypsin over a period of 12-16 h3. After removal of the ligands and residual native lactate dehydrogenase by ion exchange chromatography dimers were obtained which were almost inactive. The dimers were lacking a hexapeptide at the N-terminus; however, the secondary structure was the same as that of native lactate dehydrogenase. The circular dichroism spectra showed a dependence on temperature which suggested an equilibrium of two different structural states. The reaction of antibodies against native porcine heart LDH with the dimers restored the catalytic activity, and subsequently the dimers behaved similarly to the native enzyme. Addition of 1 M phosphate or NAD-sulfite to the dimers restored 80-90% of the catalytic activity. It could be demonstrated that the behavior of the reactivated dimers, in contrast to that of the inactive dimers, was similar to the behavior of native lactate dehydrogenase. For instance, ultracentrifugal analysis showed that dimers reactivated with NAD-SO3- were associated to give tetramers. The reaction of antibodies against native LDH with the dimers reactivated with NAD-SO3- demonstrated that the native LDH and the dimers have the same surface determinants.

Amino Acid Sequence↗
PubMed1991

Glycosylation of the Mr 46,000 mannose 6-phosphate receptor. Effect on ligand binding, stability, and conformation.

Using site-directed mutagenesis the N-glycosylation sites of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) were identified as asparagine residues 57, 83, 107, and 113. The two outer asparagines carry high mannose-type and the two inner asparagines carry complex-type oligosaccharides. The glycosylation mutants were analyzed for stability, binding activity, and subcellular distribution. Replacing asparagine 57, 83, or 107 by threonine decreased only the stability of the receptor. Replacing asparagine 113 by threonine decreased the stability and binding activity. Deletion of three or all four N-glycosylation sites led in addition to an accumulation of the mutant receptors in endoplasmic reticulum-like structures. Nonglycosylated MPR 46 synthesized in the presence of tunicamycin, thus preserving the asparagine residues, had a normal stability and high affinity binding. The decreased stability and binding activity of the receptor mutants is therefore due to the exchange of asparagine residues rather than to the loss of N-linked oligosaccharides. The nonglycosylated receptor, however, displayed a decreased conformational stability after solubilization as a single cycle of freezing and thawing reduced the binding activity to one-third of the control. Simultaneously, the receptor lost its quaternary structure. It is concluded from these results that the N-glycosylation of the receptor is required for the stability of a high affinity conformation, but not for the binding itself or the intracellular stability.

Cell Compartmentation↗
PubMed1991

Recombinant interleukin-2 in metastatic renal cell carcinoma--a European multicentre phase II study.

This multinational, multicentre study represents the introduction of recombinant interleukin-2 (rIL-2) in Europe. From December 1987 to June 1989, 57 eligible patients with metastatic renal cell cancer were treated with rIL-2 administered as continuous intravenous infusion. 8 out of 51 evaluable patients responded (16%), 2 complete remission (CR) and 6 partial remission (PR). 10 patients had no change (20%). The response duration for CR was 209 and 394+ days. The median response duration for PR was 371 (range 140-506+) days. Dose-limiting grade 3-4 toxicities were hypotension in 52% of the patients, arrhythmia (4%), dyspnoea (8%), creatinine rise (4%), peripheral neurotoxicity (10%) and central neurotoxicity (10%). Toxicities most often recovered solely on interrupted therapy. 2 patients died due to catheter-related septicaemia and one patient died of rIL-2 induced renal failure. The study confirmed the antitumour efficacy of rIL-2 in renal cell cancer. Toxicities were numerous, but manageable by close observation in a normal oncology ward without routine use of an intensive care unit.

Adult↗
PubMed1991

Molecular cloning of the mouse 46-kDa mannose 6-phosphate receptor (MPR 46).

A cDNA clone for the mouse 46-kDa mannose 6-phosphate receptor (MPR 46) was isolated from an embryonic mouse cDNA library. Its single open reading frame codes for a protein of 278 residues. It shows an over-all amino-acid identity of 93% with the human receptor. Nine non-conservative amino-acid exchanges are found in the luminal domain, one non-conservative exchange of hydrophobic amino acids is in the transmembrane domain, while the cytoplasmic receptor tails are identical. All five potential N-glycosylation sites are conserved as well as amino acids that are important for ligand binding (Arg 137 and His 131) and disulfide pairing (Cys 32 and 78, Cys 132 and Cys 167, Cys 145 and Cys 179). The absolute identity in the cytoplasmic MPR 46 tail suggests the importance of this amino-acid sequence for the intracellular routing of the MPR 46.

Amino Acid Sequence↗
PubMed1990

Hexadecylphosphocholine in the topical treatment of skin metastases in breast cancer patients.

Widespread local recurrence of breast cancer, untreatable by surgery or radiation therapy, can present a serious therapeutic problem predominantly in patients refractory to systemic therapy. In a phase I trial hexadecylphosphocholine, a new agent with high membrane affinity and antineoplastic activity was applied topically to affected skin areas of breast cancer patients. The results provide evidence that hexadecylphosphocholine may be an active agent in the topical treatment of skin metastases.

Administration, Topical↗
PubMed1989

Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding.

The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer.

Animals↗
PubMed1989

Hexadecylphosphocholine, a new ether lipid analogue. Studies on the antineoplastic activity in vitro and in vivo.

Hexadecylphosphocholine (He-PC) is a new compound synthesized according to the minimal structural requirements deducted from studies with other ether lipids. In vitro studies on He-PC revealed remarkable antineoplastic activity on HL60, U937, Raji and K562 leukemia cell lines. In addition, He-PC, applied orally, showed a superior effect in the treatment of dimethylbenzanthracene-induced rat mammary carcinomas when compared to intravenously administered cyclophosphamide. After oral application He-PC was well absorbed from the intestine and metabolized in the liver by phospholipases C and D. During a 5-week treatment no hematotoxic effects were detected. In a clinical pilot study on breast cancer patients with widespread skin involvement, topically applied He-PC showed skin tumor regressions without local or systemic side effects.

Animals↗
PubMed1988

[Results of a phase II study with the new cytostatic drug carboplatin in combination with 5-fluorouracil in the primary treatment of advanced squamous cell cancers of the head and neck].

Chemotherapeutic regimens containing Cisplatin are the most effective in the treatment of squamous cell carcinoma of the head and neck. Because of the high rate of dose-limiting side effects of Cisplatin, Carboplatin, a second generation Cisplatin analog, was tested in a phase II trial with 5-FU on 55 previously untreated patients with advanced carcinoma of the head and neck. The results of the completed study are: 33% CR, 54% PR, 10% NR and 4% PD. Toxic side effects were tolerable myelotoxicity as with Cisplatin/5FU, mild nausea, vomiting and nephrotoxicity. No ototoxicity was seen. Most patients showed better performance status after chemotherapy with increased body weight. These results indicate that Carboplatin/5-FU is more effective and has milder side effects than Cisplatin/5-FU.

Adult↗
PubMed1988

[Staging in non-Hodgkin's lymphomas].

In the period from 1970 to 1986, 46 patients with non-Hodgkin's lymphoma were laparotomized and splenectomized at the Surgery Division, University of Göttingen. The operation was frequently carried out in advanced or generalized stages with a therapeutic indication and served for purely diagnostic purposes in only a proportion of the cases. The Kiel classification was chosen for the histology. Accordingly, hairy-cell leukemia (14 cases) and chronic lymphatic leukemia (12 cases) were the most frequent diagnoses, followed by LP immunocytoma and centroblastic-centrocytic lymphoma (seven cases each). In four out of the 20 clinical pictures which were not already generalized by definition, i.e. in 20% of the cases, exploratory laparotomy resulted in a change of stage (comprising two improvements and two deteriorations). The spleen was affected in 41 out of the 46 patients; in addition, lymphomatous infiltrations were found in six of the 12 liver biopsies taken and in 15 out of 17 lymph node preparations taken. The average spleen weight in the NHL patients (1,623 g) was markedly higher than in a reference group of Hodgkin patients (317 g). With critical establishment of the indication, exploratory laparotomy with splenectomy in histologically verified NHL and lymphography in the clinical staging are the most precise methods of investigation in graduated diagnosis of non-Hodgkin's lymphomas.

Adult↗